Abstract We are studying two classes of DNA binding proteins, DNA helicases and single stranded (ss)DNA binding (SSB) proteins, that are both essential for genome maintenance in all organisms. DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA replication, recombination and repair. SSB proteins bind tightly to these ssDNA intermediates, protecting the DNA, but also bind directly to at least 17 other SSB interacting proteins (SIPs) to bring them to their sites of action. Defects in DNA helicases are responsible for a number of human diseases. We are undertaking quantitative studies of the mechanisms of DNA unwinding and ssDNA translocation of a multi-subunit DNA helicase/nuclease, E. coli RecBCD, which functions in repair of DNA double strand breaks and recombination, as well as the E. coli UvrD and Rep helicases which function in several DNA repair pathways. RecBCD is a hetero- trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (RecB, a 3' to 5' motor and RecD, a 5' to 3' motor). Despite extensive study, the mechanism of helicase DNA unwinding is not understood. There is also little known about how the two motors communicate within RecBCD and the allosteric regulation of its motor and nuclease activities by chi. We have discovered that RecBCD can unwind duplex DNA processively even in the absence of ssDNA translocation by the canonical RecB and RecD motors indicating that DNA melting and ssDNA translocation are separate processes. This ability is regulated by its nuclease and arm domains. We are studying UvrD and Rep to address the question of what is needed to turn a ssDNA translocase into a helicase and how this is regulated. By accessory proteins such as MutL and PriC. Activation of a helicase is not well understood process. E. coli SSB protein is a central player in all aspects of DNA metabolism. It can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular ssDNA binding cooperativity. A major focus is on the four intrinsically disordered C-terminal tails of SSB that we have shown regulate cooperative binding of SSB to ssDNA and control the binding of the 17 SIPs. We have developed SSB variants that selectively stabilize the different SSB binding modes and that have different numbers of C-terminal tails and different properties and will determine how these affect protein binding and DNA replication. An array of approaches, including thermodynamic, transient kinetic, structural and single molecule approaches (fluorescence and optical tweezers), will be used in these studies.